Streptococcus pyogenes strains associated with invasive and non-invasive infections present possible links with emm types and superantigens

Objective(s): Streptococcus pyogenes, a notorious human pathogen is responsible to cause a wide range of infections varies from superficial common clinical illness to severe and life threatening infections. To our knowledge this is the first report exploring the emm types and superantigen/exotoxin gene profile of S. pyogenes from Pakistan. Materials and Methods: A total of 89 S. pyogenes strains were collected predominantly from throat swabs followed by pus, tissues and wound swabs. Profile of five superantigen genes speA, speB, speC, speF and ssa was screened for all the emm types. Results: Extensive heterogeneity among S. pyogenes strains was indicated, revealing 34 different emm types/ subtypes. The most prevalent emm types were emm68 and emm104. Some of the emm types were exclusively isolated from invasive infections while others were isolated only from non-invasive infections indicating the possible link between emm types and invasive/ noninvasive infections. Similarly, erythromycin-resistant strains mainly belonged to three particular emm types. Multiplex PCR analysis indicated the presence of speB 100%, speF 76%, speC 20%, speA 18% and ssa 15%. Interestingly, superantigen genes speC and speA were mainly associated with invasive infections. Among the five superantigens tested, one strain of emm12 harbored all the analyzed exotoxin genes, while 4 strains carried 4 superantigen genes. Conclusion: S. pyogenes clones associated with invasive and non-invasive infections in Pakistan present differences in emm types, superantigens and antimicrobial resistance. The present data indicates the possible link between particular genetic lineage of a bacterium with the manifestation of the infection.


Introduction
Group A Streptococcus (GAS) associated infections occur more commonly in developing countries compared to the developed countries. Six hundred and sixteen million cases of pharyngitis, 111 million cases of pyoderma and at least 517,000 deaths due to severe invasive diseases and sequelae were reported as the global burden due to GAS diseases since 2005 (1,2). Invasive diseases are caused by the strains isolated from otherwise sterile body sites e.g. blood, cerebrospinal fluid, joints, pleural, peritoneal, or pericardial fluids or nonsterile body sites like wounds associated with streptococcal toxic shock syndrome (STSS) or necrotizing fasciitis (NF). Noninvasive GAS are isolated from patients suffering from pharyngitis, scarlet fever, erysipelas, and impetigo (3,4). STSS was first reported in the late 1980s and have been the cause of high mortality rate ranging from 30% to 70% (5-7) and NF also called as flesh eating disease involves deeper skin layers and tissues (8,9). Alarmingly, since late 1980s there has been a worldwide re-emergence of the severe forms of Streptococcus pyogenes infections, particularly NF and STSS (6,10,11).
S. pyogenes virulence factors can be divided into two groups: surface exposed virulence factors and secreted virulence factors. Among many factors involved in the pathogenesis of S. pyogenes, M protein and Streptococcal pyrogenic exotoxins (Spe) which exhibit the properties of superantigens are the most important ones (12).
M protein is a major surface virulence (and adhesion) factor of S. pyogenes and is involved in various stages of GAS pathogenesis including adhesion, internalization, immune evasion and tissue invasion. It confers resistance to phagocytosis and killing by polymorphonuclear leukocytes by binding to complement control factors and other host proteins to prevent activation of the alternate complement pathway (13)(14)(15). M protein is encoded by emm gene which exhibits marked variability in its 5' hypervariable region and forms the basis for emm genotyping (16). There are more than 100 different emm types reported to date (17). Epidemiology of virulent S. pyogenes strains from 10 countries revealed emm1, emm28, emm3, emm89, emm87, emm12, emm4, emm83, emm81 and emm5 as the most prevalent strains, particularly emm1 and emm3 were associated with STSS and NF (18). The distribution of emm gene considerably varies geographically and has been reported as an important surveillance tool for understanding the dynamics of GAS infections, its transmission, local epidemiology and indigenous vaccine development (19,20).
Among extracellular virulence factors superantigens (SAgs) are secreted proteins, which possibly contribute towards the pathogenesis of severe and invasive infections. SAgs/ exotoxins are the most potent T-cell mitogens reported so far (21). More than 40 bacterial SAgs have been reported in literature and a total of 12 antigenically distinct extracellular SAgs have been described in GAS to date which include streptococcal pyrogenic exotoxins (Spes) A, C, G-M, the streptococcal superantigen (Ssa) and streptococcal mitogenic exotoxin (SmeZ) 1 and 2 (22)(23)(24)(25). Two proteins SpeB and SpeF (also known as mitogenic factor), previously described as SAgs, infact share the properties of cysteine protease and DNase respectively and therefore are not regarded as true SAgs (26,27). SAgs are roughly 25 kDa secretory proteins that are implicated in the pathogenesis of GAS infections, including scarlet fever, STSS and rheumatic fever. SAgs have the unique ability to cross-link class II major histocompatibility complexes on antigenpresenting cells resulting in activation of up to 20% of T cells. This unspecific massive T-cell proliferation causes release of large amount of the cytokines. In addition to cytokine production and T-cell proliferation, SAgs are capable of inducing cytotoxicity towards target cells (28). These exotoxins also induce pyrogenicity, cause capillary leakage, activation of complement, coagulation and fibrinolytic cascades, leading to hypotension through cytokine release leading to shock, multiorgan failure and death. SAgs-coding genes among GAS are usually associated with bacteriophage vectors, except for speG, speJ and smeZ that are believed to be chromosomally encoded (22,29). As SAgs SpeA, SpeC, SpeH, SpeI, SpeK, SpeL, SpeM, and SSA are encoded on prophage (30), therefore can be easily transduced among strains. Certain SAgs are associated with certain emm types. Interestingly, a relationship among SAgs and emm gene types with invasive infections has been reported (31)(32)(33).
S. pyogenes was thought to be sensitive to a large number of antibiotics. Although penicillins and macrolides have remained the drugs of choice for the treatment of streptococcal infections, but in the last decade GAS drug resistant strains have been reported from worldwide (including Europe and Asia) (33)(34)(35)(36). In addition, clindamycin is considered efficient for invasive and severe streptococcal infections and fluoroquinolones can also be used, which shows the promising results during treatment (37). Many reports from developing and developed countries have shown an increase in resistance against macrolides and other antibiotics depending upon the geographical locations of the S. pyogenes strains, however only a few drug resistance reports against erythromycin have been published from Pakistan (38)(39)(40)(41).
No data was available pertaining to the minimum inhibitory concentration of antibiotics, distribution of emm types and exotoxin profile of S. pyogenes isolates from Pakistan, therefore the present study was undertaken to achieve the above objectives and to fill the gap in scientific knowledge.

Collection of S. pyogenes strains
This study was undertaken at the Department of Microbiology, University of Karachi, Pakistan. Bacterial strains and patient's basic information was obtained from different pathological laboratories, medical centres and tertiary care hospitals of Karachi. S. pyogenes strains were procured isolated from various clinical specimens like throat swabs, pus, blood, wounds, tissues, body fluids, urine and synovial fluid.

Purification and final identification of S. pyogenes strains
All the procured S. pyogenes strains were purified on sheep blood agar (to get the pure culture) and were systematically identified by the routine parameters according to Bergey's manual of determinative bacteriology diagnostic tests like catalase test, beta hemolysis, bacitracin sensitivity test etc. The confirmation of the strains was done by Lancefield grouping Kit (Oxoid, USA) while ABIS (online-advanced bacterial identification software) (www.tgw1916.net/ bacteria_logare.html) was also referred for the final identification.

Determination of minimum inhibitory concentration (MIC)
Macrodilution technique was performed using tryptone soya broth to determine MIC (42). For the interpretation of MIC, CLSI guidelines (2015) were referred for breakpoints. All the experiments were carried out in triplicate to get the average values.

Superantigen gene profile
Profile of five superantigen genes speA, speB, speC, speF and ssa was screened for all the emm types by PCR analysis with the set of primers as already described (30). Amplification of all the genes was performed with an initial 3 min denaturation at 94 o C, followed by 30 cycles of denaturation at 94 o C for 60 sec, 60 sec of annealing temperatures standardized in the laboratory for multiplex PCR and 60 sec of extension at 72 o C with a final extension step at 72 o C for 7 min.

Determination of MIC
The MIC analysis exhibited the highest level of resistance against clindamycin (1024 µg/ml) and azithromycin (512 µg/ml). Substantial level of resistance was observed for clarithromycin (128 µg/ml) and ciprofloxacin (8 ug/ml; Table 1), however all strains of S. pyogenes were found sensitive to penicillin and coamoxiclav.
Overall, 22 different emm types were found among invasive strains; among which 15 emm types were exclusively found in invasive isolates. On the other hand, noninvasive isolates comprised 19 different emm types while 12 emm types were only found in noninvasive isolates. However, 7 emm types were present in both the invasive and the noninvasive specimens. Predominantly, emm55, emm28, emm1, emm42.1 were only traced in invasive infections while emm68 was only traced in noninvasive infections. Besides, emm58, emm75, emm93, emm88.3 and emm104 were isolated from both invasive and noninvasive infections; however, emm104 was more common among noninvasive compared to the invasive infections ( Figure 1).

Superantigen gene profile
A total of 74 S. pyogenes strains were analyzed for the presence of speA, speB, speC, speF and ssa by multiplex PCR. The speB gene was present in all the strains whereas speF was present in 76% of the strains. Superantigen genes speC, speA and ssa were present in 20%, 18% and 15% of the S. pyogenes strains respectively. Interestingly, speC superantigen gene was predominantly (93%) observed in the invasive S. pyogenes strains, while speA was also found associated with 62% invasive infections (Table 2).

Discussion
To our knowledge, the present investigation is the first comprehensive study that has shed light on the prevalence, chemotherapy and pathogenesis of S. pyogenes from Pakistan. In this study, although S. pyogenes were mainly isolated from throat swabs yet almost 50% of the S. pyogenes strains importantly have been isolated from invasive specimens like pus, wound, tissues, blood, body fluids hence S. pyogenes is not only responsible for the superficial infections but equally contributes to the invasive infections in Pakistan.
Bacitracin susceptibility is used for the presumptive identification of GAS isolates, however it was observed that 16 (18%) strains of the S. pyogenes were resistant to bacitracin diagnostic disc. Similar phenomenon has also been reported from other countries (44,45).
M protein is a major surface virulence factor. Sequencing of the hypervariable region of emm gene encoding M protein has been used as the gold standard    (47)(48)(49). According to other studies the emm1, emm4, emm12 and emm28 were the predominant emm types from Austria (50), while emm 11 was reported to be the most common type from India (37,51,52).
In the current study, the emm1, emm28, emm42.1 and emm55 types were exclusively isolated from invasive infections while emm68 was only isolated from noninvasive infections thereby indicating a possible relationship between emm types and invasive/ noninvasive infections. It is known that emm1 is widely associated with streptococcal invasive diseases (49,53,54). A recent study from Japan reported the emergence of STSS associated 90% mainly with emm1, followed by emm89, emm12, emm28, emm3 and emm90 (55), while emm102 has also been associated with STSS in southern Taiwan (26). A number of similar emm types have been identified in the present study but it needs to design further comprehensive clinical, epidemiological and molecular research studies which can establish emm types association with STSS in Pakistan.
In the present study speC was significantly (93%) associated with invasive infections and speA was associated with 62% of the invasive infections. According to a previous study, emm77 was originated from invasive samples while emm1 and emm12 from noninvasive isolates (57). Although, the presence of a particular SAg gene cannot be exclusively related with the development of STSS, SAgs encoded by phage such as speA and speC have commonly been associated with invasive infections (58)(59)(60).
During the present study one strain isolated from the throat swab belonging to emm12 harbored all the five analyzed exotoxin genes, thus it can be a potentially virulent strain. Recently, it has been reported that emm12 has been associated with the emergence of scarlet fever, toxin acquisition and multidrug resistance in Hong Kong and Australia (61)(62)(63). Significant association of speA with emm1 and speC with emm12 has also been reported from Brazil (22). Similarly, 4 other strains carried 4 SAgs genes, altogether indicating the presence of virulent strains which can potentially transmit these virulent genes to other strains as well as to other groups of non-pathogenic streptococci. On the other hand the majority of strains without having SAg genes belonged to the noninvasive strains group.
The knowledge about the increased macrolide and lincosamide resistance among S. pyogenes reported by the current research study would benefit medical practioners to more accurately prescribe patients while dealing with streptococcal infections and monitoring the emergence of drug resistance.

Conclusion
As the links do exist between particular genetic lineage and the type of infection, hence the genetic makeup of the bacteria play an important role in determining the outcome of the interaction between pathogen and its host. Our data has signified the multifactorial nature of S. pyogenes strains in a particular infectious manifestation.